(1) Conventional Hydrodesulfurization Methods
Although methods such as alkali washing, solvent desulfurization etc. are known to desulfurize hydrocarbon fuels such as petroleum, hydrodesulfurization is the mainstream of desulfurization at present. Hydrodesulfurization is a method for reducing the sulfur content in a product by reacting sulfur compounds in a petroleum fraction with hydrogen in the presence of a catalyst so that they are removed as hydrogen sulfide. The catalyst used include metal catalysts such as cobalt, molybdenum, nickel, tungsten and the like carried on alumina. For a molybdenum catalyst carried on alumina, cobalt or nickel is added as a cocatalyst to improve its catalytic performance. Hydrodesulfurization using such a metal catalyst is undoubtedly a fairly completed process used widely all over the world at present. From the point of view of a process for producing petroleum products to cope with stricter environmental regulation, however, there are some problems. Hereinafter, such problems are briefly described.
Because the metal catalyst generally has relatively low substrate specificity, it is considered that this catalyst is suitable for degrading various sulfur compounds to lower the sulfur content in fossil fuels as a whole, but can be insufficient in the effect of desulfurizing a specific sulfur compound group. For example, various heterocyclic organic sulfur compounds still remain in light gas oil after such desulfurization procedures. One possible reason for such insufficient desulfurization effect of the metal catalyst is the steric hindrance caused by substituent groups around the sulfur atom in organic sulfur compounds. Among such substituent groups, the influence of methyl substituent groups on the reactivity of the metal catalyst in hydrodesulfurization has been examined using thiophene, benzothiophene, dibenzothiophene etc. According to the results, the desulfurization reactivity of the metal catalyst is decreases generally with an increasing number of substituent groups, and evidently the reactivity is also affected greatly by the position of the substituent group. It has been reported that the reactivity of the metal catalyst in desulfurizing methylated dibenzothiophenes is greatly affected by the steric hindrance caused by the substituent methyl groups (e.g. Houalla, M., Broderick, D. H., Sapre, A. V., Nag, N. K., de Beer, V. H. J., Gates, B. C., Kwart, H. J. Catalt., 61, 523-527 (1980)). In fact, a wide variety of alkylated derivatives of dibenzothiophenes are known to be present in considerable amounts in light gas oil (e.g. Kabe, T., Ishihara, A. and Tajima, H. Ind. Eng. Chem. Res., 31, 1577-1580 (1992)).
It is assumed that higher reaction temperatures or pressures than currently used are required to desulfurize the above organic sulfur compounds which are resistant to the conventional hydrodesulfurization and that a significant amount of hydrogen is also required to be added. Further, improvements in such hydrodesulfurization processes are estimated to need enormous investments in facilities and costs for operation. Such organic sulfur compounds resistant to hydrodesulfurization are contained as a major species of sulfur compounds in e.g. light gas oil. Therefore, the above-described hydrodesulfurization processes should be significantly improved in order to raise the degree of desulfurization of light gas oil.
On one hand, an enzymatic reaction carried out by organisms is characterized in that it proceeds under relatively mild conditions and the rate of an enzymatic reaction is comparable to the rate of reaction using a chemical catalyst. Furthermore, numerous enzymes are present so as to suitably deal with a wide variety of in vivo reactions, and it is known that these enzymes generally have significantly high substrate specificities. These characteristics are expected to be utilizable in microbial removal of sulfur from sulfur compounds contained in fossil fuels, that is, microbial biodesulfurization (Monticello, D. J., Hydrocarbon Processing 39-45 (1994)).
Conventional Biodesulfurization Methods:
There are a large number of reports on methods of removing sulfur from petroleum by the use of microorganisms. Joachim et al. have observed degrees of desulfurization of 60 to 80% in 2 days by continuous treatment of a highly viscous heavy oil fraction with Pseudomonas HECC39 at 30.degree. C. (Bauch, J., Herbert, G., Hieke, W., Eckart, V., Koehler, M., Babenzin, H. D., Chemical Abstracts 82530y vol. 83 (1975) ). Yuda has reported that petroleum is converted into a water-soluble compound by allowing petroleum to be in contact with Pseudomonas haconensis (Yuda, S., Unexamined Published Japanese Patent Application No. 75,107,002: Chemical Abstracts 46982j vol. 84 (1976)). In addition, Lee et al. have reported desulfurization of crude oil, light gas oil, kerosine and naphtha by a sulfur-oxidizing strain Thiobacillus thiooxidans and a sulfur-reducing strain Pseudomonas sp. (Lee, M. J., Hah, Y. C., Lee, K. W. Chemical Abstracts, 145448s, vol. 85 (1976)). They have examined the desulfurization abilities of various sulfur-oxidizing microorganisms and sulfur-reducing microorganisms, and have reported that Thiobacillus-thiooxidans has the highest ability to oxidize sulfur and Pseudomonas putrefaciens and Desulfovibrio desulfuricans have the highest ability to reduce sulfur (Lee, M. J., Hah, Y. C., Lee, K. W. Chemical Abstracts, 156414d, vol. 85 (1976)). Isolation of 7 sulfur-reducing Pseudomonas strains has also been reported by the same group. Further, Eckart et al. have reported oxidative desulfurization of Romashkino crude oil and fuel oil by Pseudomonas desmolyticum (Eckart, V., Hieke, W., Bauch, J., Gentzsch, H. Chemical Abstracts, 142230q, vol. 94 (1981); Eckart, V., Hieke, W., Bauch, J., Gentzsch, H. Chemical Abstracts, 147259c, vol. 97 (1982)). For these desulfurization reactions made by microorganisms of the genus Pseudomonas, the degradation products have been identified and it is known that every microorganism whose desulfurization reaction mechanism was revealed makes use of the cleavage reaction to C--C bonds in a sulfur compound molecule contained in oil.
(A) C--C Bond-targeted Biodesulfurization
A systematic study of microbial desulfurization was started by Yamada et al. (Yamada, K., Minoda, Y., Kodama, K., Nakatani, S., Akasaki, T., Agric. Biol. Chem.,32, 840-845 (1968)). They have reported that microorganisms of the genus Pseudomonas decompose dibenzothiophene to give a water-soluble product. The Pseudomonas strains used include Pseudomonas abikonensis and Pseudomonas jianii. Microbial desulfurization by culturing with a mixture of these 2 strains has been examined by Nakatani et al. (Nakatani, S., Sasaki, T., Kodama, K., Minoda, Y. Yamada, K., Agric. Biol. Chem. 32, 1205-1211 (1968)). In their study, a light gas oil solution containing 5% dibenzothiophene is used as a substrate. Kodama et al. have reported that amino acids or other carbon compounds are essential as cosubstrates for oxidation of benzothiophene and growth of the microorganisms. The microorganisms with desulfurization activity had the metabolism by which C--C bonds in a heterocyclic sulfur compound represented by dibenzothiophene are cleaved, the benzene rings are thereby decomposed, and via a subsequent oxidative reaction cascade, sulfates are released. The reaction mechanism of the carbon-skeleton-attack-type pathway, called the Kodama pathway, consists of hydroxylation of an aromatic ring (dibenzothiophene.fwdarw..fwdarw.1,2-dihydroxydibenzothiophene), cleavage of the ring, and oxidation thereof into a water-soluble product (1,2-dihydroxydibenzothio phene.fwdarw.trans-42-(3-hydroxy)thianenaphthenyl!-2-oxo-butenoic acid, 3-hydroxy-2-formylbenzothiophene). Although this type of reaction is known in the genus Pseudomonas, it has been confirmed that the degradation reaction of dibenzothiophene by this kind of microorganism is catalyzed by the same enzymatic group as that participating in naphthalene degradation (Eaton, R. W. and Chapman, P. J., J. Bacteriol., 174, 7542-7554, 1992; Denome, S. A., Stanley, D. C., Olson, E. S. and Young, K. D., J. Bacteriol., 175, 6890-6901, 1993). The studies on the microorganisms revealed the microbial ability to remove dibenzothiophene and substituted dibenzothiophene from a pentane-soluble fraction separated from crude oil. From one such strain Pseudomonas alcaligenes (DBT-2), 25 kb DNA involved in oxidation of dibenzothiophene was isolated and cloned in a multiple copy expression vector (Finnerty, W. R. and Robinson, M., Biotechnol., Bioengineer. Symp. #16, 205-221 (1986)). In these cases, C--C bonds in the benzene ring of dibenzothiophene are attacked and various water-soluble substances capable of extraction from oil are formed. During this reaction, however, other aromatic molecules in oil are also attacked and as a result a significant amount of hydrocarbons move to the liquid phase (Hartdegen, F. J., Coburn, J. M. and Roberts, R. L., Chem. Eng. Progress, 80, 63-67 (1984)). Such reaction leads to a reduction in the total thermal unit in petroleum and is thus an industrially unacceptable reaction. Further, this type of microorganism for oxidative degradation of dibenzothiophene gives a water-soluble thiophene compound (mainly 3-hydroxy-2-formylbenzothiophene) as the oxidation product as reported by Kodama et al., which is, however, a substance difficult to remove from the aqueous phase.
Besides said microorganisms, certain microorganisms are known to attack a carbon skeleton in the same manner as above, to catalyze partial oxidation of organic sulfur heterocyclic compounds and to convert them into water-soluble products; examples of such microorganisms are Pseudomonas sp.,Pseudomonas aeruginosa, Beijerinckia sp., Pseudomonas alcaligenes, Pseudomonas stutzeri and Pseudomonas putida (which catalyze partial oxidation) and Brevibacterium sp. (which catalyzes mineralization i.e. mineral formation). The genetically determinative elements of these enzymatic reactions, which represent bio-transformations unique to oxidation of aromatic hydrocarbons, are believed to be generally carried on plasmids (Monticell, D. J., Bakker, D., Finnerty, W. R. Appl. Environ. Microbiol, 49, 756-760 (1985)). The enzymatic reactions in these microbial systems are not of sulfur-targeted types and are thus not functional for removing organic sulfur from high-molecular-weight fractions separated from crude oil, and the usefulness of the microorganisms in bio-processing of fossil fuels having a high content of sulfur is therefore limited. The reasons for this are: (1) attack on the carbon ring of dibenzothiophene occurs often at the 2- and 3-positions of dibenzothiophenes substituted with alkyl or allyl groups at those positions, and the dibenzothiophenes substituted at those positions do not serve as substrates in the Kodama pathway; (2) the pathway for destroying the carbon skeleton reduces the energy content of fuel; and (3) the major product of the pathway for destroying the carbon skeleton is 3-hydroxy-2-formylbenzothiophene, while a trace amount of dibenzothiophene is decomposed to form a sulfate, so that adequate desulfurization does not occur.
(B) C--S Bond-Targeted Type Biodesulfurization
There are reports of microorganisms degrading not only crude oil and coal but also model compounds containing sulfur so that sulfur is selectively removed as a heteroatom and sulfates and hydroxide compounds are produced. From the structures of their metabolites, these types of reactions are considered to be reactions in which C--S bonds in sulfur compounds are specifically cleaved and as a result the sulfur is released in the form of sulfate. Aerobic and heterotrophic non-acidophilic soil microorganisms Pseudomonas CB1 and Acinetobacter CB2 were reported to convert thiophene sulfur into sulfate (Isbister, J. D. and Kobylinski, E. A. Microbial desulfurization of coal. in Coal Science and Technology, Ser. 9, p. 627 (1985). When a bench-scale continuous bioreactor was used, the content of organic sulfur in Illinois #6 coal was reduced 47% by use of CB1. Dibenzothiophene sulfoxide, dibenzothiophene sulfone, and 2,2'-dihydroxybiphenyl have been identified as intermediates of dibenzothiophene in desulfurization. Separately, it has been reported that 35 to 45% of the organic sulfur content in 4 different types of coal is removed as sulfates by unidentified microorganisms isolated from soil (Finnerty, W. R. and Robinson, M., Biotechnol. Bioengineer. Symp. #16, 205-221 (1986)). In addition, an isolated strain, Rhodococcus rhodochrous ATCC53968, has a sulfur-targeted-type pathway for converting dibenzothiophene into hydroxybiphenyl and sulfate, and it is said that 70% of the organic sulfur content in crude oil and coal is reduced by this microorganism (Kilbane, J. J. Resources, Conservation and Recycling, 3, 69-70 (1990)). For Corynebacterium sp., there is also a description of a pathway for degrading dibenzothiophene by similarly oxidizing dibenzothiophene and converting it via dibenzothiophene sulfoxide, then dibenzothiophene sulfone, into 2-hydroxybiphenyl and sulfate (Ohmori, T., Monna, L., Saiki, Y. and Kodama, T. Appl. Environ. Microbiol., 58, 911-915, 1992). In this case, the 2-hydroxybiphenyl is further converted into nitrates to form 2 different hydroxy nitrobiphenyls. Recently, there are also reports on oxidation of dibenzothiophene into benzoic acid and nitrite by Brevibacterium sp. Do (van Afferden. M., Schacht, S., Klein, J. and Truper, H. G. Arch. Microbiol., 153, 324-328, 1990) and oxidation of benzyl methyl sulfide into benzaldehyde by Pseudomonas sp. OS1 (van Afferden, M., Tappe, D., Beyer, M., Truper, H. G. and Klein, J. Fuel 72, 1635-1643, 1993). Arthrobacter K3b has been reported to exhibit a reaction similar to that of the Brevibacterium, and when dibenzothiophene sulfone is used as a substrate, sulfite and benzoic acid are produced (Dahiberg, M. D. (1992) Third International Symposium on the Biological Processing of Coal, May 4-7, Clearwater Beach, Fla., pp. 1-10, Electric Power Research Institute, Palo Alto, Calif.). Meanwhile, a novel system has also been reported in which conversion of a sulfur-containing aromatic heterocyclic compound into hydrogen sulfide is carried out in a non-aqueous solvent (Finnerty, W. R. Fuel 72, 1631-1634, 1993). An unidentified strain FE-9 converts dibenzothiophene into biphenyl and hydrogen sulfide in 100% dimethylformamide in a hydrogen atmosphere or into hydroxybiphenyl and sulfate in the presence of air. This strain is further reported to convert thianthrene into benzene and hydrogen sulfide in a hydrogen atmosphere or into benzene and sulfates in the presence of air. Besides such microorganisms aerobically degrading dibenzothiophene, anaerobic and sulfate-reducing microorganisms are also reported to convert dibenzothiophene into biphenyl and hydrogen sulfide and to convert petroleum organic sulfur biologically into hydrogen sulfide (Kim, H. Y., Kim, T. S. and Kim, B. H., Biotechnol. Lett. 12, 757-760, 1990a; Kim, T. S., Kim, H. Y. and Kim, B. H., Biotechnol. Lett. 12, 761-764, 1990b). The C--S bond-targeted type biodesulfurizing microorganisms as described above are summarized as follows:
TABLE 1 __________________________________________________________________________ C--S Bond Attack Type Microorganisms Strain Substrate Degradation Product Literature __________________________________________________________________________ Pseudomonas sp. CB1 dibenzothiophene; hydroxybiphenyl + Isbister et al. coal sulfate (1985) Acinetobacter sp. CB2 dibenzothiophene hydroxybiphenyl + Isbister et al. sulfate (1985) Grain-positive coal sulfate Crawford et al. bacteria (1990) Rhodococcus rhodochrous dibenzothiophene; hydroxybiphenyl + Kilbane IGTS8 (ATCC 53968) coal, petroleum sulfate (1989) Desulfovibrio dibenzothiophene biphenyl + Kim et al. desulfuricans hydrogen sulfide (1990) Corynebacterium sp. dibenzothiophene hydroxybiphenyl + Omori et al. sulfate (1992) Brevibacterium sp. DC dibenzothiophene benzoic acid + van Alferden et al. sulfite (1990) Gram-positive dibenzothiophene; biphenyl + Finnerty bacterium FE-9 thianthrene hydrogen sulfide (1993) benzene + hydrogen sulfide Pseudomonas sp. OS1 benzylmethylsulfide benzaldehyde van Afferden (1993) Rhodococcus dibenzothiophene hydroxybiphenyl Wang et al. erythropolis (1994) Rhodococcus dibenzo- hydroxy- Izumi et al. (1994) erythropolis thiophene biphenyl Ohshiro et al. (1995) D-1, H-2 Agrobacterium sp. dibenzothiophene hydroxybiphenyl Constanti et al. (1994) Xanthomanas sp. dibenzothiophene hydroxybiphenyl Constanti et al. (1994) Arthrobacter K3b dibenzothiophene benzoic acid + Dahlberg sulfite (1992) __________________________________________________________________________
(C) Conventional High-Temperature Biodesulfurization Methods
Microbial metabolic reactions proceeding at a temperatures of about 30.degree. C. are utilized in every biodesulfurization described above. On the other hand, it is known that the rate of chemical reaction increases generally depending on temperature. In the desulfurization step in a petroleum refining process, fractional distillation and desulfurization reactions are carried out under high-temperature and high-pressure conditions. For incorporating a biodesulfurization step into a petroleum refining process, therefore, it is considered desirable that the biodesulfurization reaction be carried out at higher temperature during cooling, without cooling a petroleum fraction to normal temperature. There are the following reports on high-temperature biodesulfurization.
Most of the attempts at desulfurization by microorganisms at high temperature can be found in coal desulfurization. A variety of sulfur compounds are contained in coal. The major inorganic sulfur compound is iron pyrite, while the organic sulfur compound is present in the form of a mixture of a wide variety of organic sulfur compounds, many of which are known to contain thiol, sulfide, disulfide and thiophene groups. The microorganisms used are those of the genus Sulfolobus, all of which are thermophilic microorganisms. There are reports in which a wide variety of Sulfolobus strains have been used for the leaching of metals from mineral sulfide (Brierley C. L. & Murr, L. E., Science 179, 448-490 (1973)) and removal of sulfur from iron pyrite in coal (Kargo, F. & Robinson, J. M., Biotechnol. Bioeng. 24, 2115-2121 (1982); Kargi, F. & Robinson, J. M., Appl. Environ. Microbiol., 44, 878-883 (1982); Kargi, F. & Gervoni. T. D., Biotechnol. Letters 5, 33-38 (1983); Kargi, F. and Robinson, J. M., Biotechnol. Bioeng., 26, 687-690 (1984); Kargi, F. & Robinson, J. M., Biotechnol. Bioeng. 27, 41-49 (1985); Kargi, F., Biotechnol. Lett., 9, 478-482 (1987)). According to Kargi and Robinson (Kargi. F. and Robinson, J. M., Appl. Environ. Microbiol., 44, 878-883 (1982)), a certain strain of Sulfolobus acidocaldarius isolated from an acidic hot spring in the Yellowstone National Park, US, grows at 45 to 70.degree. C. and oxidizes elementary sulfur optimally at pH 2. Further, oxidation of iron pyrite by two other Sulfolobus acidocaldarius strains has also been reported (Tobita, M., Yokozeki, M., Nishikawa, N. & Kawakami, Y., Biosci. Biotech. Biochem. 58, 771-772 (1994)).
Among the organic sulfur compounds contained in fossil fuels, dibenzothiophene and its substituted derivatives are known to hardly undergo hydrodesulfurization in a petroleum refining process. High-temperature degradation of dibenzothiophene by Sulfolobus acidocaldarius has also been reported (Kargi, K. & Robinson, J. M., Biotechnol. Bioeng. 26, 687-690 (1984); Kargi, F., Biotechnol. Letters 9, 478-482 (1987)). According to these reports, when model aromatic heterocyclic sulfur compounds such as thianthrene, thioxanthene and dibenzothiophene were reacted at high temperature with this microorganism, these sulfur compounds were oxidized and decomposed. The oxidation of these aromatic heterocyclic sulfur compounds by S. acidocaldarius has been observed at 70.degree. C., and sulfate ion is formed as the reaction product. However, this reaction was carried out in a medium not containing a carbon source except for the sulfur compound. Therefore, this sulfur compound can also be used as a carbon source by this organism. That is, C--C bonds in the sulfur compound are evidently decomposed. Further, this microorganism Sulfolobus acidocaldarius can grow only in an acidic medium, so the oxidative degradation of dibenzothiophene by this microorganism is required to proceed under severe acidic conditions (pH 2.5). Such severe conditions are considered undesirable for the process because deterioration of petroleum products are caused under such conditions and simultaneously acid-resistant materials are required for the desulfurization step. If Sulfolobus acidocaldarius is allowed to grow under autotrophic conditions, it derives necessary energy from reduced iron and sulfur compounds and utilizes carbon dioxide as a carbon source. If Sulfolobus acidocaldarius is allowed to grow under heterotrophic conditions, it can utilize a wide variety of organic compounds as both carbon and nitrogen sources. Namely, if fossil fuels are present, these would be assimilated as a carbon source by the microorganism.
Finnerty et al. have reported that strains belonging to Pseudomonas stutzeri, Pseudomonas alcaligenes and Pseudomonas putida decompose dibenzothiophene, benzothiophene, thioxanthene and thianthrene to convert them into water-soluble substances (Finnerty, W. R., Shockiey, K., Attaway, H. in Microbial Enhanced Oil Recovery, Zajic, J. E. et al. (eds.) Penwell. Tulsa, Okla., 83-91 (1983)). The oxidation reactions in these cases are assumed to proceed even at 55.degree. C. However, the degradation product of dibenzothiophene by these Pseudomonas strains was 3-hydroxy-2-formylbenzothiophene as reported by Kodama et al. (Monticello, D. J., Bakker, D., Finnerty, W. R. Appl. Environ. Microbiol., 49, 756-760 (1985)). The activity of these Pseudomonas strains in oxidizing dibenzothiophene is induced by naphthalene or salicylic acid i.e. a sulfur-free aromatic hydrocarbon while inhibited by chloramphenicol. As can be seen from this, the degradation reaction of dibenzothiophene by the Pseudomonas strains is based on the degradation by cleavage of C--C bonds in an aromatic ring. Thus, their degradation can occur not only in sulfur compounds but also in important aromatic hydrocarbons contained in petroleum fractions, resulting in reduction in the value of the fuel as well as the qualities of the petroleum fractions.
The microorganisms so far found to be capable of degrading dibenzothiophene at high temperatures catalyze cleavage of C--C bonds in a dibenzothiophene molecule and utilize it as a carbon source. As described above, the degradation reactions of organic sulfur compounds, in which C--S bonds are specifically cleaved while C--C bonds are not cleaved and remain, are desirable for actual petroleum desulfurization methods as discussed above (Conventional Biodesulfurization Methods). That is, the use of microorganisms having the activity of cleaving C--S bonds in dibenzothiophene and its alkyl substituted derivatives at high temperatures and forming desulfurization products in the form of water-soluble substances is most preferable for the biodesulfurization process.
As described above, microorganisms in some genera are known to exhibit C--S-bond-cleavage-type degradation reactions against dibenzothiophene. However, there is no description that such microorganisms demonstrated the activity of degrading dibenzothiophene under high-temperature conditions at 42.degree. C. or more. For example, Rhodococcus sp. ATCC 53968 is a well studied dibenzothiophene-degrading strain, and by the reaction of this microorganism, an oxygen atom is added to the sulfur atom of dibenzothiophene, and the resulting dibenzothiophene sulfoxide is converted into dibenzothiophene sulfone which via 2'-hydroxybiphenyl-2-sulfinate, is further converted into 2-hydroxybiphenyl. However, the growth of this microorganism in a 48 hour culture is also significantly delayed or prevented at 43.degree. C. or even at 37.degree. C. slightly higher than the usual culture temperature of 30.degree. C. (Unexamined Published Japanese Patent Application No. 6-54695). It is therefore considered the most suitable for high-temperature desulfurization to use a microorganism capable of growing at high temperatures and degrading organic sulfur compounds, particularly heterocyclic sulfur compounds such as dibenzothiophene and its substituted derivatives by specifically cleaving C--S bonds in said compounds.